Jitsuhiro Matsugi

Biochemical properties of Caenorhabditis elegans HMG-5, a regulator of mitochondrial DNA

Caenorhabditis elegans HMG-5, which is encoded by F45E4.9, contains two high mobility group (HMG) box domains and shows sequence similarity with mammalian mitochondrial transcription factor A (TFAM). In this study, using soaking RNA interference, we found that knockdown of HMG-5 reduced the amount of mtDNA in P0 hermaphrodites, suggesting it as functional orthologue of mammalian TFAM. We also examined the biochemical property of HMG-5 in mammalian cells and in vitro. We found that HMG-5 localized to the mitochondria in human cultured cells and was included in the NP-40-insoluble fraction in which mtDNA and TFAM were enriched. By immunoprecipitation analysis, HMG-5 was found to associate with human mitochondrial DNA (mtDNA) in the cells. In vitro binding experiment also showed that HMG-5 binds to C. elegans mtDNA and plasmid DNA, indicating its feature as a non-specific DNA-binding protein. Furthermore, it was found that HMG-5 can interact with itself. These results demonstrate that HMG5 shares similar biochemical properties with mammalian TFAM as a nucleoid factor. HMG-5 could be a good candidate for investigating mtDNA metabolism in multicellular organisms.

Nucleotide sequences of serine tRNAs from Bacillus subtilis

Three B. subitilis serine tRNAs were sequenced including modified nucleosides. All the serine tRNAs contained 1-methyl-adenosine in the D-loop. As other characteristic modified nucleosides, 5-methoxyuridine was found in the first letter of the anticodon in the tRNA(UGA).

Intracellular NF-HEV/IL-33 harbors essential roles in Ras-induced cellular transformation by contributing to cyclin D1 protein synthesis

A member of the interleukin-1 family, interleukin-33 (NF-HEV/IL-33), is a ligand for the receptor, ST2L and stimulates the production of Th2 cytokines. Although IL-33 localizes to the nucleus and may be involved in the regulation of transcription independent of ST2L, its functions in the nucleus currently remain unclear. We herein demonstrated that the expression of IL-33 was markedly enhanced in NIH-3T3 cells transformed by an oncogenic H-Ras mutant (H-Ras (G12V)), and the induced IL-33 was mainly located in the nuclei of these cells. The enforced expression of IL-33 accelerated H-Ras (G12V)-induced transformation in NIH-3T3 cells, and this transforming activity was markedly reduced by the knockdown of IL-33 with shRNA. We subsequently analyzed several signaling molecules regulated by Ras in order to elucidate the mechanism by which IL-33 contributes to Ras (G12V)-induced transformation. We found that the knockdown of IL-33 effectively attenuated the Ras (G12V)-induced expression of cyclin D1. However, the knockdown of IL-33 failed to affect cyclin D1 mRNA expression levels, and epoxomicin, a proteasome inhibitor, did not cancel the IL-33 knockdown-induced down-regulation of its protein levels. We showed that Ras (G12V)-induced cyclin D1 protein synthesis was markedly suppressed by the knockdown of IL-33. Taken together, the results of the present study strongly suggest a novel role for IL-33 in cellular transformation.

STAT3 and ERK pathways are involved in cell growth stimulation of the ST2/IL1RL1 promoter

The ST2 gene was originally identified as a primary responsive gene induced by stimulation with growth factors and by oncogenic stress. The ST2 gene harbors two distinct promoters – a distal promoter and a proximal promoter. In this study, we identified a novel type of serum‐responsive element in the ST2 proximal promoter using reporter gene analysis; this element includes a possible responsive element for STAT family proteins. Indeed, enforced expression of constitutively active STAT3 activated this promoter element and induced the expression of ST2 gene products. Furthermore, an oncogenic Ras (G12V) mutant also caused the expression of ST2 gene products by utilizing the proximal promoter. We also clarified that activation of the ST2 promoter by either growth stimulation or oncogenic Ras was suppressed by the inhibitors for STAT3 and ERK pathways. Our observations strongly suggest the importance of STAT family and ERK pathways for the induction of ST2 gene products by cell growth stimulation.

ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

The ST2 gene was originally identified as a primary responsive gene, and the expressions of its gene products are induced by stimulation with growth factors and by oncogenic stresses. In this study, we observed that oncogenic Ras mutant induced the expression of ST2 and ST2L proteins. Interestingly, the enforced expression of ST2 gene products in NIH-3T3 murine fibroblasts remarkably enhanced Ras (G12V)-induced cellular transformation. Furthermore, when the expression of ST2 gene products was silenced by RNA-interference technique, Ras (G12V)-induced cellular transformation was drastically suppressed. According to these observations, it was indicated that the oncogenic Ras-induced expression of ST2 gene products is required for the acceleration of cellular transformation, and this seems to be independent of the stimulation with IL-33, a ligand for ST2/ST2L. Interestingly, knockdown of ST2 gene products caused a reduction in Rb phosphorylation in transformed murine fibroblasts, suggesting the functional involvement of ST2 gene products in cell cycle progression during cellular transformation. Our current study strongly suggests the importance of ST2 gene products in cellular transformation, and the presence of novel mechanism how ST2 gene products affect the cellular transformation and cell proliferation.